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Thursday, 18 December 2014

Like human patients, mice with a form of Duchenne muscular dystrophy undergo progressive muscle degeneration and accumulate connective tissue as they age. Now, researchers at the Stanford University School of Medicine have found that the fault may lie at least partly in the stem cells that surround the muscle fibres.

They've found that during the course of the disease, the stem cells become less able to make new muscle and instead begin to express genes involved in the formation of connective tissue. Excess connective tissue – a condition called fibrosis – can accumulate in many organs, including the lungs, liver and heart, in many different disorders. In the skeletal muscles of people with muscular dystrophy, the fibrotic tissue impairs the function of the muscle fibres and leads to increasing weakness and stiffness, which are hallmarks of the disease.

The researchers discovered that this abnormal change in stem cells could be inhibited in laboratory mice by giving the animals a drug that is already approved for use in humans. The drug works by blocking a signalling pathway involved in the development of fibrosis. Although much more research is needed, the scientists are hopeful that a similar approach may one day work in children with muscular dystrophy.

"These cells are losing their ability to produce muscle, and are beginning to look more like fibroblasts, which secrete connective tissue," said Thomas Rando, MD, PhD, professor of neurology and neurological sciences.

"It's possible that if we could prevent this transition in the muscle stem cells, we could slow or ameliorate the fibrosis seen in muscular dystrophy in humans."

A paper describing the researchers' findings will be published Dec. 17 in Science Translational Medicine. Rando, the paper's senior author, is director of the Glenn Laboratories for the Biology of Aging and founding director of the Muscular Dystrophy Association Clinic at Stanford. Former postdoctoral scholar Stefano Biressi, PhD, is the lead author. Biressi is now at the Centre for Integrative Biology at the University of Trento in Italy.

A devastating disease

Duchenne muscular dystrophy is a devastating disease that affects about 1 in every 3,600 boys born in the United States. Patients usually experience severe, progressive muscle weakness that confines them to a wheelchair in early adolescence and eventually leads to paralysis. It's caused by mutations in the dystrophin gene, which encodes the dystrophin protein. The dystrophin protein serves to connect muscle fibres to the surrounding external matrix. This connection stabilizes the fibres, enhancing their strength and preventing injury. Sufferers are nearly always boys because the dystrophin gene is located on the X chromosome. (Girls would need to inherit two faulty copies, which is unlikely because male carriers often die in early adulthood.)

Under normal conditions, muscle stem cells respond to muscle damage by dividing into cells, one of which becomes new muscle, while the other remains a stem cell. However, in the mice missing the dystrophin gene, the muscle stem cells slowly assume a different fate. They begin to resemble fibroblasts instead of muscle-making machines.

To conduct the research, Biressi and Rando used a strain of laboratory mice in which the muscle stem cells were engineered to glow with a fluorescent light when treated with a drug called tamoxifen. They then bred the mice with another strain in which the dystrophin gene is mutated, and followed the fate of the cells over time.

The researchers found that the expression of myogenic genes, which are associated with the regeneration of muscle in response to injury, was nearly completely lacking in many of the muscle stem cells in the mice after just 11 months, while the expression of fibrotic genes had increased compared with that of control animals. The cells from the dystrophic animals were also oddly located: Rather than being nestled next to the muscle fibres, they had begun to move away into the spaces between tissues.

The role of a signalling pathway

Rando and Biressi knew that a similar, but much less pronounced, accumulation of connective tissue in muscle fibres occurs during normal aging. That process is governed by signalling proteins, which include the Wnt and TGF-beta protein families. Wnt plays a critical role in embryonic development and cancer; TGF-beta controls cell division and specialization. They wondered whether blocking the Wnt/TGF-beta pathway in the dystrophic mice would inhibit fibrosis in the animals' muscles.

The researchers turned to a drug called losartan, which is used to treat high blood pressure. Losartan inhibits the expression of the genes for TGF-beta types 1 and 2. The researchers thought it would probably interrupt the signalling pathway that leads the muscle stem cells astray.

Treating the mice with losartan, they found, did in fact prevent the muscle stem cells from expressing fibrosis-associated genes and partially maintained their ability to form new muscle.

"So we'd like to understand why it happens, and how to prevent it. It's also important to limit fibrosis to increase the likelihood of success with other possible therapies, such as cell therapy or gene therapy."

Next steps

Because TGF-beta type 1 plays many roles throughout the body, the researchers are now working to find ways to specifically inhibit TGF-beta type 2, which is involved in the transition of the muscle stem cells. They're also interested in learning how to translate the research to other diseases.

"Fibrosis seems to occur in a vicious cycle," Rando said.

"As the muscle stem cells become less able to regenerate new muscle, the tissue is less able to repair itself after damage. This leads to fibrosis, which then further impairs muscle formation. Understanding the biological basis of fibrosis could have a profound effect on many other diseases."

As the main component of connective tissue in the body, fibroblasts are the most common type of cell. Taking advantage of that ready availability, scientists from the Perelman School of Medicine at the University of Pennsylvania, the Wistar Institute, Boston University School of Medicine, and New Jersey Institute of Technology have discovered a way to repurpose fibroblasts into functional melanocytes, the body's pigment-producing cells. The technique has immediate and important implications for developing new cell-based treatments for skin diseases such as vitiligo, as well as new screening strategies for melanoma. The work was published this week in Nature Communications.

"Through direct reprogramming, we do not have to go through the pluripotent stem cell stage, but directly convert fibroblasts to melanocytes. So these cells do not have tumourigenicity."

Changing a cell from one type to another is hardly unusual. Nature does it all the time, most notably as cells divide and differentiate themselves into various types as an organism grows from an embryo into a fully-functional being. With stem cell therapies, medicine is learning how to tap into such cell specialization for new clinical treatments. But controlling and directing the process is challenging. It is difficult to identify the specific transcription factors needed to create a desired cell type. Also, the necessary process of first changing a cell into an induced pluripotent stem cell (iPSC) capable of differentiation, and then into the desired type, can inadvertently create tumours.

Xu and his colleagues began by conducting an extensive literature search to identify 10 specific cell transcription factors important for melanocyte development. They then performed a transcription factor screening assay and found three transcription factors out of those 10 that are required for melanocytes: SOX10, MITF, and PAX3, a combination dubbed SMP3.

"We did a huge amount of work," says Xu.

"We eliminated all the combinations of the other transcription factors and found that these three are essential."

The researchers first tested the SMP3 combination in mouse embryonic fibroblasts, which then quickly displayed melanocytic markers. Their next step used a human-derived SMP3 combination in human foetal dermal cells, and again melanocytes (human-induced melanocytes, or hiMels) rapidly appeared. Further testing confirmed that these hiMels indeed functioned as normal melanocytes, not only in cell culture but also in whole animals, using a hair-patch assay, in which the hiMels generated melanin pigment. The hiMels proved to be functionally identical in every respect to normal melanocytes.

Xu and his colleagues anticipate using their new technique in the treatment of a wide variety of skin diseases, particularly those such as vitiligo for which cell-based therapies are the best and most efficient approach.

The method could also provide a new way to study melanoma.

By generating melanocytes from the fibroblasts of melanoma patients, Xu explains: "we can screen not only to find why these patients easily develop melanoma, but possibly use their cells to screen for small compounds that can prevent melanoma from happening."

Perhaps most significantly, say the researchers, is the far greater number of fibroblasts available in the body for reprogramming compared to tissue-specific adult stem cells, which makes this new technique well-suited for other cell-based treatments.

Monday, 15 December 2014

Consider the relationship between an air traffic controller and a pilot. The pilot gets the passengers to their destination, but the air traffic controller decides when the plane can take off and when it must wait. The same relationship plays out at the cellular level in animals, including humans. A region of an animal's genome - the controller - directs when a particular gene - the pilot - can perform its prescribed function.

These are
images of mouse embryonic stem cells

which grow
in a round colony of cells (A) and

express
Sox2 (B), shown in red. Sox2 control

region-deleted
cells have lost the typical

appearance
of embryonic stem cells (C) and do

not express
Sox2 (D). The DNA is shown in blue

in B and D.
Credit: Jennifer Mitchell/University

of Toronto.

A new study by cell and systems biologists at the University of Toronto (U of T) investigating stem cells in mice shows, for the first time, an instance of such a relationship between the Sox2 gene which is critical for early development, and a region elsewhere on the genome that effectively regulates its activity. The discovery could mean a significant advance in the emerging field of human regenerative medicine, as the Sox2 gene is essential for maintaining embryonic stem cells that can develop into any cell type of a mature animal.

"We studied how the Sox2 gene is turned on in mice, and found the region of the genome that is needed to turn the gene on in embryonic stem cells," said Professor Jennifer Mitchell of U of T's Department of Cell and Systems Biology, lead investigator of a study published in the December 15 issue of Genes & Development.

"Like the gene itself, this region of the genome enables these stem cells to maintain their ability to become any type of cell, a property known as pluripotency. We named the region of the genome that we discovered the Sox2 control region, or SCR," said Mitchell.

Since the sequencing of the human genome was completed in 2003, researchers have been trying to figure out which parts of the genome made some people more likely to develop certain diseases. They have found that the answers are more often in the regions of the human genome that turn genes on and off.

"If we want to understand how genes are turned on and off, we need to know where the sequences that perform this function are located in the genome," said Mitchell.

"The parts of the human genome linked to complex diseases such as heart disease, cancer and neurological disorders can often be far away from the genes they regulate, so it can be difficult to figure out which gene is being affected and ultimately causing the disease."

It was previously thought that regions much closer to the Sox2 gene were the ones that turned it on in embryonic stem cells. Mitchell and her colleagues eliminated this possibility when they deleted these nearby regions in the genome of mice and found there was no impact on the gene's ability to be turned on in embryonic stem cells.

"We then focused on the region we've since named the SCR as my work had shown that it can contact the Sox2 gene from its location 100,000 base pairs away," said study lead author Harry Zhou, a former graduate student in Mitchell's lab, now a student at U of T's Faculty of Medicine.

"To contact the gene, the DNA makes a loop that brings the SCR close to the gene itself only in embryonic stem cells. Once we had a good idea that this region could be acting on the Sox2 gene, we removed the region from the genome and monitored the effect on Sox2."

The researchers discovered that this region is required to both turn Sox2 on, and for the embryonic stem cells to maintain their characteristic appearance and ability to differentiate into all the cell types of the adult organism.

"Just as deletion of the Sox2 gene causes the very early embryo to die, it is likely that an abnormality in the regulatory region would also cause early embryonic death before any of the organs have even formed," said Mitchell.

"It is possible that the formation of the loop needed to make contact with the Sox2 gene is an important final step in the process by which researchers practicing regenerative medicine can generate pluripotent cells from adult cells."

"Though the degree to which human embryonic stem cells possess this feature is not entirely clear, by understanding how another complex organism's genome works we ultimately learn more about how our own genome works," said Zhou.

Scientists at McMaster University have discovered that human stem cells made from adult donor cells "remember" where they came from and that's what they prefer to become again.

This means the type of cell obtained from an individual patient to make pluripotent stem cells, determines what can be best done with them. For example, to repair the lung of a patient with lung disease, it is best to start off with a lung cell to make the therapeutic stem cells to treat the disease, or a breast cell for the regeneration of tissue for breast cancer patients.

Pluripotency is the ability stem cells have to turn into any one of the 226 cell types that make up the human body. The work challenges the previously accepted thought that any pluripotent human stem cell could be used to similarly generate the same amount of mature tissue cells.

This finding, published today in the prestigious science journal Nature Communications, will be used to further drug development at McMaster, and potentially improve transplants using human stem cell sources.

"It's like the stem cell we make wants to become a doctor like its grandpa or an artist like its great-grandma," said Bhatia.

"We've shown that human induced pluripotent stem cells, called iPSCs, have a memory that is engraved at the molecular/genetic level of the cell type used to make them, which increases their ability to differentiate to the parent tissue type after being put in various stem cell states.”

"So, not all human iPSCs are made equal," Bhatia added.

"Moving forward, this means that iPSC generation from a specific tissue requiring regeneration is a better approach for future cellular therapies. Besides being faster and more cost-efficient in the development of stem cell therapy treatments, this provides a new opportunity for use of iPSCs in disease modelling and personalized drug discovery that was not appreciated before."

Neural cells generated from
patient-specific stem cells.

Small quantities of tissue would need to be harvested from the patient; for example, a few millilitres of blood or a small skin punch biopsy. From there, the harvested cells can be put into various stem cell states where they make copies of themselves indefinitely. The trickier part has been to get them out of copy function model and into differentiation mode to produce the cells needed for researchers to test drug therapies with no harm to patients, said Bhatia.

The McMaster Stem Cell and Cancer Research Institute is the only Canadian university team continuing to work exclusively with the more fragile human stem cells rather than mouse stem cells for its research, and that has furthered the work to future clinical impact. By contrast, the iPSCs of mice, which are widely used in stem cell research, have no memory, the authors note.

"So, if you only studied the mouse alone, you'd never uncover this opportunity," said Bhatia.

In a previous discovery, Bhatia and his team discovered how to make human blood from adult human skin. This meant that patients needing blood would be able to have blood created from a patch of their own skin to provide transfusions.

With these new findings, "our starting block has changed," said Bhatia, adding that now researchers "can make better tailored human stem cells for therapies because we've got a more efficient way of making higher quality and quantity of cells. For example, our team has shown that the stem cells that come from blood in the first place make blood 10 times better."

Researchers at the Center for iPS Cell Research and Application (CiRA), Kyoto University, show that induced pluripotent stem (iPS) cells can be used to correct genetic mutations that cause Duchenne muscular dystrophy (DMD). The research, published in Stem Cell Reports, demonstrates how engineered nucleases, such as TALEN and CRISPR, can be used to edit the genome of iPS cells generated from the skin cells of a DMD patient. The cells were then differentiated into skeletal muscles, in which the mutation responsible for DMD had disappeared.

DMD is a severe muscular degenerative disease caused by a loss-of-function mutation in the dystrophin gene. It inflicts 1 in 3500 boys and normally leads to death by early adulthood. Currently, very little is available in terms of treatment for patients outside palliative care. One option gaining interest is genomic editing by TALEN and CRISPR, which have quickly become invaluable tools in molecular biology. These enzymes allow scientists to cleave genes at specific locations and then modify the remnants to produce a genomic sequence to their liking. However, programmable nucleases are not pristine and often mistakenly edit similar sequences that vary a few base pairs from the target sequence, making them unreliable for clinical use because of the potential for undesired mutations.

For this reason, induced pluripotent stem cells (iPS cells) are ideal models, because they provide researchers an abundance of patient cells on which to test the programmable nucleases and find optimal conditions that minimize off-target modifications. CiRA scientists took advantage of this feature by generating iPS cells from a DMD patient. They used several different TALEN and CRISPR to modify the genome of the iPS cells, which were then differentiated into skeletal muscle cells. In all cases, dystrophin protein expression was convalesced, and in some cases, the dystrophin gene was fully corrected.

One key to the success was the development of a computational protocol that minimized the risk of off-target editing. The team built a database that all possible permutations of sequences up to 16 base pairs long. Among these, they extracted those that only appear once in the human genome, i.e. unique sequences. DMD can be caused by several different mutations; in the case of the patient used in this study, it was the result of the deletion of exon 44. The researchers therefore built a histogram of unique sequences that appeared in a genomic region that contained this exon. They found a stack of unique sequences in exon 45.

to Akitsu Hotta, who headed the project and holds joint positions at CiRA and the Institute for Integrated Cell-Materials Sciences at Kyoto University:

"Nearly half the human genome consists of repeated sequences. So even if we found one unique sequence, a change of one or two base pairs may result in these other repeated sequences, which risks the TALEN or CRISPR editing an incorrect region. To avoid this problem, we sought a region that hit high in the histogram".

With this target, the team considered three strategies to modify the frame-shift mutation of the dystrophin gene: exon skipping by connecting exons 43 and 46 to restore the reading frame, frame shifting by incorporating insertion or deletion (indel) mutations, and exon knock-in by inserting exon 44 before exon 45. All three strategies effectively increased dystrophin synthesis in differentiated skeletal cells, but only the exon knock-in approach recovered the gene to its natural state. Importantly, editing showed very high specificity, suggesting that their computational approach can be used to minimize off-target editing by the programming nucleases.

Moreover, the paper provides a proof-of-principle for using iPS cell technology to treat DMD in combination with TALEN or CRISPR. The group now aims to expand this protocol to other diseases.

First author Lisa Li explains: "We show that TALEN and CRISPR can be used to correct the mutation of the DMD gene. I want to apply the nucleases to correct mutations for other genetic-based diseases like point mutations".

Friday, 21 November 2014

While investigating a rare genetic disorder, researchers at the University of California, San Diego School of Medicine have discovered that a ubiquitous signalling molecule is crucial to cellular reprogramming, a finding with significant implications for stem cell-based regenerative medicine, wound repair therapies and potential cancer treatments.

The findings are published in the Nov. 20 online issue of Cell Reports.

Karl Willert, PhD, assistant professor in the Department of Cellular and Molecular Medicine, and colleagues were attempting to use induced pluripotent stem cells (iPSC) to create a "disease-in-a-dish" model for focal dermal hypoplasia (FDH), a rare inherited disorder caused by mutations in a gene called PORCN. Study co-authors V. Reid Sutton and Ignatia Van den Veyver at Baylor College of Medicine had published the observation that PORCN mutations underlie FDH in humans in 2007.

FDH is characterized by skin abnormalities such as streaks of very thin skin or different shades, clusters of visible veins and wart like growths. Many individuals with FDH also suffer from hand and foot abnormalities and distinct facial features. The condition is also known as Goltz syndrome after Robert Goltz, who first described it in the 1960s. Goltz spent the last portion of his career as a professor at UC San Diego School of Medicine. He retired in 2004 and passed away earlier this year.

To their surprise, Willert and colleagues discovered that attempts to reprogram FDH fibroblasts or skin cells with the requisite PORCN mutation into iPSCs failed using standard methods, but succeeded when they added WNT proteins - a family of highly conserved signalling molecules that regulate cell-to-cell interactions during embryogenesis.

"WNT signalling is ubiquitous," said Willert.

"Every cell expresses one or more WNT genes and every cell is able to receive WNT signals. Individual cells in a dish can grow and divide without WNT, but in an organism, WNT is critical for cell-cell communication so that cells distinguish themselves from neighbours and thus generate distinct tissues, organs and body parts."

WNT signalling is also critical in limb regeneration (in some organisms) and tissue repair.

"We've shown that WNT signalling is required for cellular reprogramming," said Willert.

"Some of the processes that occur during cellular reprogramming resemble those that occur during regenerative processes and wound repair. For example, limb regeneration in organisms like axolotl and zebrafish require cells at the injury site to de-differentiate (change their function) and then rebuild the damaged tissue. WNT is essential for these amazing regenerative processes."

Willert cautioned that "it would be a stretch to say that activation of WNT signalling will allow us to regenerate limbs," but said WNT activation is likely valuable in assisting tissue repair.

A variety of efforts are already underway exploring how to leverage WNT signalling to promote wound healing, such as speeding bone fracture repairs, and even hair growth.

"That's not really a wound repair process, but WNT is required for hair growth," Willert said.

The caveat, he noted, is that "there's a fine line between repairing tissue and promoting cancer growth." Willert said there are efforts underway to create therapeutics that block WNT signalling as a means to block cancer growth. Earlier this year, for example, Willert and colleagues published findings describing the use of an antibody to disrupt WNT signalling in embryonic stem cells. In cancer cells with mutations in the WNT signalling pathway this antibody may inhibit their growth and development.

Pluripotent Cells Created by Nuclear Transfer Can Prompt Immune Reaction

Friday, 21 November 2014

Mouse cells and tissues created through nuclear transfer can be rejected by the body because of a previously unknown immune response to the cell's mitochondria, according to a study in mice by researchers at the Stanford University School of Medicine and colleagues in Germany, England and at MIT.

The findings reveal a likely, but surmountable, hurdle if such therapies are ever used in humans, the researchers said.

Stem cell therapies hold vast potential for repairing organs and treating disease. The greatest hope rests on the potential of pluripotent stem cells, which can become nearly any kind of cell in the body. One method of creating pluripotent stem cells is called somatic cell nuclear transfer, and involves taking the nucleus of an adult cell and injecting it into an egg cell from which the nucleus has been removed.

The promise of the SCNT method is that the nucleus of a patient's skin cell, for example, could be used to create pluripotent cells that might be able to repair a part of that patient's body.

"One attraction of SCNT has always been that the genetic identity of the new pluripotent cell would be the same as the patient's, since the transplanted nucleus carries the patient's DNA," said cardiothoracic surgeon Sonja Schrepfer, MD, PhD, a co-senior author of the study, which will be published online Nov. 20 in Cell Stem Cell.

"The hope has been that this would eliminate the problem of the patient's immune system attacking the pluripotent cells as foreign tissue, which is a problem with most organs and tissues when they are transplanted from one patient to another," added Schrepfer, who is a visiting scholar at Stanford's Cardiovascular Institute. She is also a Heisenberg Professor of the German Research Foundation at the University Heart Center in Hamburg, and at the German Center for Cardiovascular Research.

Possibility of rejection

A dozen years ago, when Irving Weissman, MD, professor of pathology and of developmental biology at Stanford, headed a National Academy of Sciences panel on stem cells, he raised the possibility that the immune system of a patient who received SCNT-derived cells might still react against the cells' mitochondria, which act as the energy factories for the cell and have their own DNA. This reaction could occur because cells created through SCNT contain mitochondria from the egg donor and not from the patient, and therefore could still look like foreign tissue to the recipient's immune system, said Weissman, the other co-senior author of the paper. Weissman is the Virginia and D.K. Ludwig Professor for Clinical Investigation in Cancer Research and the director of the Stanford Institute for Stem Cell Biology and Regenerative Medicine.

That hypothesis was never tested until Schrepfer and her colleagues took up the challenge.

"There was a thought that because the mitochondria were on the inside of the cell, they would not be exposed to the host's immune system," Schrepfer said.

"We found out that this was not the case."

Schrepfer, who heads the Transplant and Stem Cell Immunobiology Laboratory in Hamburg, used cells that were created by transferring the nuclei of adult mouse cells into enucleated eggs cells from genetically different mice. When transplanted back into the nucleus donor strain, the cells were rejected although there were only two single nucleotide substitutions in the mitochondrial DNA of these SCNT-derived cells compared to that of the nucleus donor.

"We were surprised to find that just two small differences in the mitochondrial DNA were enough to cause an immune reaction," she said.

"We didn't do the experiment in humans, but we assume the same sort of reaction could occur," Schrepfer added.

Until recently, researchers were able to perform SCNT in many species, but not in humans. When scientists at the Oregon Health and Science University announced success in performing SCNT with human cells last year, it reignited interest in eventually using the technique for human therapies. Although many stem cell researchers are focused on a different method of creating pluripotent stem cells, called induced pluripotent stem cells, there may be some applications for which SCNT-derived pluripotent cells are better suited.

Handling the reaction

The immunological reactions reported in the new paper will be a consideration if clinicians ever use SCNT-derived stem cells in human therapy, but such reactions should not prevent their use, Weissman said.

"This research informs us of the margin of safety that would be required if, in the distant future, we need to use SCNT to create pluripotent cells to treat someone," he said.

"In that case, clinicians would likely be able to handle the immunological reaction using the immunosuppression methods that are currently available."

In the future, scientists might also lessen the immune reaction by using eggs from someone who is genetically similar to the recipient, such as a mother or sister, Schrepfer added.

Tuesday, 18 November 2014

Milestone for understanding diseases and for the development of new therapies

Tuesday, 18 November 2014

Scientists at Hiroshima University established induced pluripotent stem (iPS) cells from the fibroblasts of Werner Syndrome patients. These results were published in PLOS ONE in an article entitled "Reprogramming Suppresses Premature Senescence Phenotypes of Werner Syndrome Cells and Maintains Chromosomal Stability over Long-Term Culture."

Werner syndrome is characterized by the premature appearance of features associated with normal aging and cancer predisposition. This syndrome occurs frequently in Japan, affecting 1 in 20,000 to 1 in 40,000 people. The therapeutic methods for this disease are very limited and it is expected that iPS cells can be used for the development of innovative therapies.

Dr. Akira Shimamoto and his collaborators analysed patient-derived iPS cells and found that telomeric abnormalities in the fibroblasts of these patients, which were caused by the lack of WRN helicase encoded by the gene responsible for Werner syndrome, were recovered in the iPS cells generated from these patients. Furthermore, Dr. Shimamoto found that the expression levels of aging-related genes, including those encoding cell cycle inhibitors and inflammatory cytokines, in the patient-derived iPS cells were the same as those in normal iPS cells, even though the expression levels of these genes in the fibroblasts of the patients were higher than those in normal fibroblasts.

"So far, the use of patient cells was restricted to blood or dermal cells in basic research. The iPS cells that we have established will provide an opportunity for drug discovery for the treatment of Werner syndrome and also help with better understanding of the mechanism of this disease. In addition, the mutated WRN gene in patient-derived iPS cells can be corrected by genome editing. This advantage will be help in the development of new gene and cell therapies for Werner syndrome," Dr. Shimamoto said.

Associate Professor Akira Shimamoto and Professor Hidetoshi Tahara at the Graduate School of Biomedical & Health Science in Hiroshima University, Professor Koutaro Yokote at the Graduate School of Medicine in Chiba University, Visiting Professor Makoto Goto at the Medical Center East in Tokyo Women's Medical University, and collaborators including the staff at the Cancer Chemotherapy Center in the Japanese Foundation for Cancer Research, Tottori University, and Keio University also participated in the study.

Researchers at the Cedars-Sinai Heart Institute have found that injections of cardiac stem cells might help reverse heart damage caused by Duchenne muscular dystrophy, potentially resulting in a longer life expectancy for patients with the chronic muscle-wasting disease.

The study results were presented today at a Breaking Basic Science presentation during the American Heart Association Scientific Sessions in Chicago. After laboratory mice with Duchenne muscular dystrophy were infused with cardiac stem cells, the mice showed steady, marked improvement in heart function and increased exercise capacity.

Duchenne muscular dystrophy, which affects 1 in 3,600 boys, is a neuromuscular disease caused by a shortage of a protein called dystrophin, leading to progressive muscle weakness. Most Duchenne patients lose their ability to walk by age 12. Average life expectancy is about 25. The cause of death often is heart failure because the dystrophin deficiency leads to cardiomyopathy, a weakness of the heart muscle that makes the heart less able to pump blood and maintain a regular rhythm.

"Most research into treatments for Duchenne muscular dystrophy patients has focused on the skeletal muscle aspects of the disease, but more often than not, the cause of death has been the heart failure that affects Duchenne patients," said Eduardo Marbán, MD, PhD, director of the Cedars-Sinai Heart Institute and study leader.

"Currently, there is no treatment to address the loss of functional heart muscle in these patients."

During the past five years, the Cedars-Sinai Heart Institute has become a world leader in studying the use of stem cells to regenerate heart muscle in patients who have had heart attacks. In 2009, Marbán and his team completed the world's first procedure in which a patient's own heart tissue was used to grow specialized heart stem cells. The specialized cells were then injected back into the patient's heart in an effort to repair and regrow healthy muscle in a heart that had been injured by a heart attack. Results, published in The Lancet in 2012, showed that one year after receiving the experimental stem cell treatment, heart attack patients demonstrated a significant reduction in the size of the scar left on the heart muscle.

Earlier this year, Heart Institute researchers began a new study, called ALLSTAR, in which heart attack patients are being infused with allogeneic stem cells, which are derived from donor-quality hearts.

Recently, the Heart Institute opened the nation's first Regenerative Medicine Clinic, designed to match heart and vascular disease patients with appropriate stem cell clinical trials being conducted at Cedars-Sinai and other institutions.

In the study, 78 lab mice were injected with cardiac stem cells. Over the next three months, the lab mice demonstrated improved pumping ability and exercise capacity in addition to a reduction in heart inflammation. The researchers also discovered that the stem cells work indirectly, by secreting tiny fat droplets called exosomes. The exosomes, when purified and administered alone, reproduce the key benefits of the cardiac stem cells.

Marbán said the procedure could be ready for testing in human clinical studies as soon as next year. The process to grow cardiac-derived stem cells was developed by Marbán when he was on the faculty of Johns Hopkins University. Johns Hopkins has filed for a patent on that intellectual property and has licensed it to Capricor, a company in which Cedars-Sinai and Marbán have a financial interest. Capricor is providing funds for the ALLSTAR clinical trial at Cedars-Sinai.

The Cedars-Sinai Heart Institute has been at the forefront of developing investigational stem cell treatments for heart attack patients.

Harvard Stem Cell Institute (HSCI) researchers, representing five Harvard departments and affiliated institutions as well as the Massachusetts Institute of Technology (MIT), have demonstrated that adult cells, reprogrammed into another cell type in a living animal, can remain functional over a long period.

Joe Zhou is a Harvard Stem Cell Institute

Principal Faculty Member and an Associate

Professor in Harvard's Department of Stem Cell

and Regenerative Biology. Zhou and colleagues

have demonstrated that cells reprogrammed in

vivo can survive, thrive, and be
therapeutically

useful. Credit: B.
D. Colen/Harvard University.

The work by Joe Zhou, an associate professor in Harvard's Department of Stem Cell and Regenerative Biology, and his collaborators is an important advance in the effort to develop cell-based therapies for tissue repair, and specifically in the effort to develop improved treatment for diabetes.

The researchers used a combination of genes to change pancreatic exocrine cells – one of the main forms of cells in the pancreas – in adult mice that have diabetes into insulin-producing beta cells that appeared to cure about a third of the mice of the metabolic disease, and improved insulin production in most of the other mice. A report on the work was published today in the journal Nature Biotechnology.

The new findings are a major advance in work by HSCI co-director Doug Melton and Zhou, who in 2008 reported having converted exocrine cells into functional beta cells in mice. At that time, however, it was not known how long, and how well, the repurposed cells would function.

"The efficiency of reprogramming has always been an issue," Zhou said.

"Until now, the new cells have either dropped dramatically in number or disappeared completely," he said, noting that since his work with Melton in 2008 there have been reports published in other programing systems that question whether the reprogrammed cells could be stable enough ultimately to be useful.

"What we have demonstrated is that yes, the reprogrammed cells can be useful, and for that to happen you have to create a niche environment in which the cells can survive," Zhou continued.

"We have improved the reprogramming efficiency to a point where one can create a large enough number of the new cells that the new cells create their own niche environment."

Zhou said that the researchers studied the mice for up to about 13 months, approximately half their normal life span, and found that "the cells are still there, and fairly robust. These are diabetic animals, and we were able to, I wouldn't use the word 'cure' because that's a very freighted word for me to use, but they became highly glycaemic animals – though not every animal became normal. That may be because to completely control the glucose level of the animal, you not only need beta cells, you need about a quarter of a million functional beta cells. If you are short of this number, even if the beta cells are perfectly normal," they can't completely control blood sugar levels, Zhou said.

When discussing the implications of the study for the field of cellular reprogramming, Zhou cautioned that the pancreas has a particularly simple cellular organization and structure, and thus findings in the pancreas might not necessarily apply to other organs.

Diabetes is a metabolic disease that is seen in two basic forms.

Type 1 diabetes is an autoimmune disease affecting about 3 million Americans, in which the patient's immune system ultimate destroys all the insulin-producing beta cells in the pancreas, and the patient has to inject insulin in order to regulate blood glucose levels.

Type 2 diabetes, which is now at epidemic prevalence rates in the United States and around the world, is usually caused by being overweight, lack of proper exercise, and improper diet, and can make a patient insulin-resistant, so the insulin the body produces is not sufficient to control blood glucose levels.

If the kind of treatment approach suggested by the new study were to succeed in humans – and that is a question to be answered with further animal, and eventually human, studies – it could be useful in treating both forms of diabetes.

One drawback to the current form of the new approach is that the cellular reprogramming is done with genes, and there might ultimately be unwanted effects on the cells. Zhou said the goal would be to replace the genes with chemicals or, perhaps, RNAs.

"I've talked to many clinicians about whether our approach could be used in humans," Zhou said.

"And the opinion is divided. Some say this could be developed into a human treatment, and some say it should be improved. But there seems to be general agreement that it could potentially be useful."